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Whole Genome DNA extraction from Tetrahymena
Day 1 Materials *''Tetrahymena'' stock cultures *Petridish(es) *SPP medium Method #Inoculate 15 mL 1 x SPP medium (+ Penicillin G & Streptomycin sulfate + Amphotericin) in a standard-sized petridish with 250 μL of healthy stock of culture. #Incubate at 30 °C in a moisture chamber until cells are stationary (usually 24 hours). Day 2 Materials *Log-phase cultures of Tetrahymena *Water bath (set at 55 C) *NDS+Pronase solution Make NDS + Pronase solution just before use. 600 uL of solution is required for each 15 mL of Tetrahymena culture. #Carefully pour each culture into a 15 mL sterile conical centrifuge-tube and centrifuge at 600 g for 3 minutes. #While the cultures are beign cerifuged, pipette 300 uL of NDS+Pronase solution into microcentrifuge tubes (use 2 microcentrifuge tubes per 15 mL culture) and place in the 55 C water bath. Addition of EDTA to SDS may cause it to precipitate. Incubate NDS solution at 55 C for a few minutes before adding Pronase solution as this will cause the SDS to go back into solution. '' #Pour off supernatant, giving the tubes a quick tap to leave about 0.2 mL cell pellet. Re-suspend the pellet in the remaining fluid by flicking the tube. #Using large orifice pipette tips, add 100 uL of cell suspension to each of the labeled microcentrifuge tubes (containing NDS+Pronase). #Pipette to mix well while avoiding bubble formation. #Incubate tubes overnight in the 55 C water bath. Start extractions the next day or store at 4 C for later use. Day 3 Materials *TE solution (1 M Tris pH 8, 0.5 M EDTA) *Phenol *Chisam (1 part isoamyl alcohol: 24 parts chloroform) *100% ethanol chilled *70% ethanol chilled ''The Phenol:Chisam solution should be colorless; phenol solutions in Tris buffers will turn yellow eventually upon oxidation and should be discarded. '' Method ''All steps should be performed under a chemical hood. Phenol is caustic and can cause serious chemical burns. It is rapidly absorbed through the skin and inhaling vapors may have anesthetic effects and fatal. Exercise extreme caution when working with chloroform too. #Begin extractions by adding 300 μL of TE to one tube from each culture. Store the other tube at 4 °C as a backup. You can extract it when and if you need more DNA. #Extract once with 700 μL of a 1:1 mixture of phenol and “chisam”. There are 2 layers in the bottle; the phenol/chisam is the bottom layer. #Mix by inverting tubes three or four times. Centrifuge at 12,000 rpm for 4 minutes. Remove top aqueous layer with large orifice tip and transfer to a new labeled microcentrifuge tube. #Extract this top layer with 700 μL chisam. Mix by inverting tubes three or four times. Centrifuge at 12,000 rpm for 4 minutes. Remove top aqueous layer with large orifice tip and transfer to a new labeled microcentrifuge tube. #Add 1 mL ice cold 100% ethanol to each tube to precipitate DNA. Mix well by inverting tubes; a cloudy precipitate should form. This is the DNA. #Centrifuge at 12000 rpm for 15 minutes. Pour off supernatant and leave pellet at bottom of tube. #Rinse pellet with 0.5 mL 70% ethanol. Spin at 12,000 rpm for 30 seconds. Pour off ethanol (to decrease chances of losing pellet). #Rinse pellet with 0.5 mL of 100% ethanol. Spin and pour off ethanol. #Dry pellets in the hood (~30 minutes). #Add 400 μL TE to each pellet. Shake ON at 200 rpm at room temperature to make the DNA go into solution. Day 4 Materials *Acétate de sodium (NaOAc) 3M pH7.. *TE solution (1M Tris pH8, 0.5 M EDTA) *Ice cold 100% ethanol *Ice cold 70% ethanol Method #Re-precipitate DNA by adding 40 μL of 3M NaOAc and 1 mL ice cold 100% ethanol. Mix by inverting tubes. You should see a cloudy precipitate. #Spin tubes for 15 minutes at 12,000 rpm. Pour off supernatant and leave pellet at bottom of tube. #Rinse pellet with 0.5 ml of 70% ethanol. Spin 30 seconds at 12,000 rpm. Pour off ethanol to decrease chances of losing pellet. #Rinse pellet with 0.5 ml of 100% ethanol. Spin and pour off ethanol. #Dry pellet in the hood (about half an hour). #Add 200 μL of TE to each pellet. Shake ON at 200 rpm at RT (DNA will go into solution). Day 5 Materials *RNase A (30 mg/mL) MethodAdd 1 μL RNase A to each tube. Incubate at 37 °C for 2 hours. Quantitate DNA by method of choice. Expect DNA concentrations in range of 200-800 ng/μl. #Store DNA at -20 °C. Category:Tetrahymena Category:DNA Category:Extraction Category:Whole Genome